The detection of mechanical stimuli by hair cells, the sensory receptors cells of the inner ear, underlies our senses of hearing and balance. Despite substantial research efforts, the molecular basis of hair-cell mechanotransduction is poorly understood. In particular, little progress has been made in the identification and characterization of the proteins that constitute the hair-cell transduction machinery. Substantial data indicate that one protein of the vestibular hair-cell transduction machinery has been identified. This protein, an unconventional myosin called myosin-1 c, serves as the motor protein that powers slow adaptation, which maintains the hair cell's sensitivity to small stimuli. In its role as the adaptation motor, myosin-lc must interact with the other components of the transduction machinery including the elusive mechanically-gated transduction channel. Our long-term goal is to understand how myosin-lc and other hair-cell transduction proteins assemble into a mechanosensitive complex and how they function in our senses of hearing and balance. The objective of this application is to extend our recent studies that examined the interaction of myosin-lc with proteins located at stereociliary tips, the site of hair-cell transduction and to exploit this interaction to isolate other components of the transduction apparatus. Our central hypothesis is that the neck domain of myosin-1 c binds to constituents of the transduction apparatus and that myosin-1 c's calmodulin light chains regulate this interaction, in addition to modulating the myosin's motor activity. In this proposal we will (1) define the portion of the myosin-lc neck responsible for binding to other transduction components; (2) examine the role of the neck domain in myosin-1 c motor activity and (3) use the myosin-lc neck region as a tool to identify and characterize other transduction components. These studies will provide insight into the molecular basis of hair-cell mechanotransduction, which is critical for an understanding of how we sense both sound and head position.